The development of cost-effective methods for the conversion of lignocellulose to ethanol offers many potential opportunities to solve environmental waste problems and to replace petroleum-based automotive fuels (Hohmann, et al., 1993; Sheehan, 1994). However, it is difficult to produce high ethanol concentrations from most lignocellulosic biomass.
Recently, recombinant microorganisms have been developed that produce higher ethanol concentrations from lignocellulose relative to other microorganisms. For example, E. coli KO11 is a recombinant derivative of E. coli B in which the Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) have been integrated into the host chromosome (Ohta et al., 1991 and U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; and 5,482,846). Genetically engineered bacteria, such as Escherichia coli KO11, have been shown to be effective biocatalysts for the fermentation of hemicellulose hydrolysates (Beall et al., 1992) and mixed waste office paper (Brooks and Ingram, 1995), achieving ethanol levels of 40-50 gL.sup.-1 within 48 to 72 h (Katzen and Fowler, 1994). However, to achieve these levels of ethanol production, fermenting and processing of up to 20 L of broth per liter of ethanol is required. Thus, there is a need for an inexpensive nutrient media to make ethanol production from lignocellulosic biomass economically feasible.
As with yeast (Jones et al., 1994; Thomas et al., 1990), the rate of fermentation and ethanol yield with ethanologenic bacteria are higher when complex nutrients are added (Lawford et al., 1991). Amino nitrogen has been identified as being of particular importance (Guimaraes et al., 1992; Jones et al., 1994a; Thomas et al., 1990). Additionally, organic nitrogen, amino acids, and commercial proteases are marketed as supplements for the yeast-based ethanol industries (Jones et al., 1994b). However, commercial, purified protein hydrolysates are too costly ($3.50-$18.00 kg.sup.-1 in bulk) for ethanol production.
Corn steep liquor is widely used as an inexpensive microbial nutrient supplement (Atkinson and Mavituna, 1991) and supports excellent ethanol production by the pentose-fermenting yeast Pichia stipitis (Amartey and Jeffries, 1994) and E. coli KO11 (Asghari et al., 1996). Corn steep liquor, however, has a number of problems, including a lack of availability in some areas and variations in quality (Shah and Cheryan, 1995). Most importantly, corn steep liquor contains toxins which require removal prior to use. The removal of toxins adds additional steps and expense in the production of ethanol. Additionally, the variations in quality mean that additional testing must be conducted each time a new batch of corn steep liquor is used so that the corn steep liquor can be optimized in conjunction with the other components to maximize ethanol production.